Asthma is characterized by airway hyperreactivity and remodeling, and chronic inflammation of the conducting airways. A critical event in the inflammatory response with the airways is the epithelial activation of nuclear factor ?B (NF-?B), a transcription factor that regulates the expression of many genes involved in the inflammatory process. Induction of nitric oxide (NO) synthase is a common feature of airway inflammation, and various cell studies have illustrated inhibitory effects of NO"""""""" on NF ?B mediated gene expression, presumably by S-nitrosation of critical proteins. Since inflammatory conditions involve recruitment of granulocytes and activation of oxidant-producing enzymes, such as eosinophil peroxidase (EPO), metabolism of NO"""""""" is likely to be altered because of oxidative conversion to potentially proinflammatory reactive nitrogen species (RNS), and bioactivity of NO"""""""" (through S-nitrosation) may be reduced. Indeed, subnormal S-nitrosothiol levels have been found in airway secretions of severe asthmatics, and formation of RNS has been demonstrated by increased tyrosine nitration. We therefore hypothesize that increased oxidative NO"""""""" metabolism results in reduced S-nitrosation of components of the NF-?B pathway, thereby promoting its chronic activation and consequently augmenting airway inflammation. Addressing such a hypothesis has been difficult because of a lack of adequate tools to detect S-nitrosoproteins in intact cells or tissues. We have adapted a recently developed procedure, based on chemical derivatization to selectively biotinylate and/or purify S-nitrosated proteins, allowing us to detect S-nitrosated proteins in intact tissue sections and to collect S-nitrosated proteins for identification of cellular targets by more global proteomic analysis. We plan to use these approaches to determine changes in S-nitrosation in relation to NO metabolism in a mouse model of allergic airway inflammation (Aim 1), and to identify changes in S-nitrosation in specific proteins in relation to alterations in NF-?B activation and gene expression, in cultured airway epithelial cells (Aim 2) and in airway of mice with allergic airway inflammation (Aim 3). Finally, with the use of various (knock-out) models of deficiency in EPO or myeloperoxidase (MPO) and of overexpression of catalase, we plan to explore a role of oxidant-producing enzymes in alterations in NO"""""""" metabolism, and in changes in S-nitrosation of proteins involved in NF-?B activation and gene expression. Collectively, we anticipate that these studies will provide causal links between NO"""""""" metabolism, NF-?B activation, and chronic airway inflammation. ? ?
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